Development of In Vitro Macrophage System to Evaluate Phagocytosis and Intracellular Fate of Penicillium marneffei Conidia

Penicillium marneffei is a pathogenic fungus that can cause a life-threatening systemic mycosis in the immunocompromised hosts. We established the model for the phagocytosis of P. marneffei conidia by RAW264.7 murine macrophages and designated the fate of P. marneffei in RAW264.7 cells with respect to persistence, phagosome–lysosome-fusion. And we impaired the immune status of mouse and compared the fate and phagosome–lysosome-fusion of P. marneffei in immunocompetent and immunosuppressed mouse peritoneal macrophages cells. We found that conidia could germinate and survive in macrophages. Within 30 min and up to 2 h of heat-killed conidia internalization, the majority of all phagosome types were labeled for the EEA1 (endosomal markers) and LAMP-1 (lysosomal markers), respectively. But both the percentages of LAMP-1 and EEA1 that associated with live conidia were significantly lower than that with heat-killed conidia. Administration of cyclophosphamide resulted in a significant suppression of macrophages function (phagocytic and fungicidal) against P. marneffei that were not apparently seen. Our data provide the evidence that (i) intracellular conversion of P. marneffei conidia into yeast cells still could be observed in macrophages. (ii) Phagosomes containing live Penicillium marneffei conidia might inhibit the phagosome–lysosome-fusion and result to no acidification surrounding the organisms. (iii) Immunity impaired by cyclophosphamide could not influence the function, including phagocytosis, fungicidal activity and phagosome–lysosome-fusion, of macrophages against P. marneffei.     

Mp1p Is a Virulence Factor in Talaromyces (Penicillium) marneffei

BackgroundTalaromyces marneffei is an opportunistic dimorphic fungus prevalent in Southeast Asia. We previously demonstrated that Mp1p is an immunogenic surface and secretory mannoprotein of T. marneffei. Since Mp1p is a surface protein that can generate protective immunity, we hypothesized that Mp1p and/or its homologs are virulence factors.Conclusions/SignificanceMp1p is a key virulence factor of T. marneffei. Mp1p mediates virulence by improving the survival of T. marneffei in macrophages.     

An inexpensive point-of-care immunochromatographic test for Talaromyces marneffei infection based on the yeast phase specific monoclonal antibody 4D1 and Galanthus nivalis agglutinin

Talaromyces marneffei is a thermally dimorphic fungus that causes opportunistic systemic mycoses in patients with AIDS or other immunodeficiency syndromes. The purpose of this study was to develop an immunochromatographic strip test (ICT) based on a solid phase sandwich format immunoassay for the detection of T. marneffei antigens in clinical urine specimens. The T. marneffei yeast phase specific monoclonal antibody 4D1 (MAb4D1) conjugated with colloidal gold nanoparticle was used as a specific signal reporter. Galanthus nivalis Agglutinin (GNA) was adsorbed onto nitrocellulose membrane to serve as the test line. Similarly, a control line was created above the test line by immobilization of rabbit anti-mouse IgG. The immobilized GNA served as capturing molecule and as non-immune mediated anti-terminal mannose of T. marneffei antigenic mannoprotein. The MAb4D1–GNA based ICT showed specific binding activity with yeast phase antigen of T. marneffei, and it did not react with other common pathogenic fungal antigens. The limit of detection of this ICT for T. marneffei antigen spiked in normal urine was approximately 0.6 μg/ml. The diagnostic performance of the ICT was validated using 341 urine samples from patents with culture- confirmed T. marneffei infection and from a control group of healthy individuals and patients with other infections in an endemic area. The ICT exhibited 89.47% sensitivity, 100% specificity, and 97.65% accuracy. Our results demonstrate that the urine-based GNA–MAb4D1 based ICT produces a visual result within 30 minutes and that the test is highly specific for the diagnosis of T. marneffei infection. The findings validate the deployment of the ICT for clinical use.     

In Vivo Yeast Cell Morphogenesis Is Regulated by a p21-Activated Kinase in the Human Pathogen Penicillium marneffei

Pathogens have developed diverse strategies to infect their hosts and evade the host defense systems. Many pathogens reside within host phagocytic cells, thus evading much of the host immune system. For dimorphic fungal pathogens which grow in a multicellular hyphal form, a central attribute which facilitates growth inside host cells without rapid killing is the capacity to switch from the hyphal growth form to a unicellular yeast form. Blocking this transition abolishes or severely reduces pathogenicity. Host body temperature (37°C) is the most common inducer of the hyphal to yeast transition in vitro for many dimorphic fungi, and it is often assumed that this is the inducer in vivo. This work describes the identification and analysis of a new pathway involved in sensing the environment inside a host cell by a dimorphic fungal pathogen, Penicillium marneffei. The pakB gene, encoding a p21-activated kinase, defines this pathway and operates independently of known effectors in P. marneffei. Expression of pakB is upregulated in P. marneffei yeast cells isolated from macrophages but absent from in vitro cultured yeast cells produced at 37°C. Deletion of pakB leads to a failure to produce yeast cells inside macrophages but no effect in vitro at 37°C. Loss of pakB also leads to the inappropriate production of yeast cells at 25°C in vitro, and the mechanism underlying this requires the activity of the central regulator of asexual development. The data shows that this new pathway is central to eliciting the appropriate morphogenetic response by the pathogen to the host environment independently of the common temperature signal, thus clearly separating the temperature- and intracellular-dependent signaling systems.     

Caenorhabditis elegans: A Simple Nematode Infection Model for Penicillium marneffei

Penicillium marneffei, one of the most important thermal dimorphic fungi, is a severe threat to the life of immunocompromised patients. However, the pathogenic mechanisms of P. marneffei remain largely unknown. In this work, we developed a model host by using nematode Caenorhabditis elegans to investigate the virulence of P. marneffei. Using two P. marneffei clinical isolate strains 570 and 486, we revealed that in both liquid and solid media, the ingestion of live P. marneffei was lethal to C. elegans (P<0.001). Meanwhile, our results showed that the strain 570, which can produce red pigment, had stronger pathogenicity in C. elegans than the strain 486, which can’t produce red pigment (P<0.001). Microscopy showed the formation of red pigment and hyphae within C. elegans after incubation with P. marneffei for 4 h, which are supposed to be two contributors in nematodes killing. In addition, we used C. elegans as an in vivo model to evaluate different antifungal agents against P. marneffei, and found that antifungal agents including amphotericin B, terbinafine, fluconazole, itraconazole and voriconazole successfully prolonged the survival of nematodesinfected by P. marneffei. Overall, this alternative model host can provide us an easy tool to study the virulence of P. marneffei and screen antifungal agents.     

Functional identification of the DHN melanin synthesis gene cluster and its role in UV-C tolerance in citrus postharvest pathogenic fungus Penicillium digitatum

The filamentous fungus Penicillium digitatum brings out great losses in citrus fruits by causing citrus green mold disease during the postharvest period. Previously, we obtained a T-DNA insertion mutant N2130 of P. digitatum, which produced albino conidia. To understand the role of green-grey conidial pigment in P. digitatum, we identified the insertion site and deeply explored the 1,8-dihydroxynaphtsalene (DHN)-melanin synthesis gene cluster in this phytopathogen. In this study, we deleted five genes in P. digitatum, PdPksP, PdAbr1, PdArp1, PdArp2, and PdAyg1, and the experiments were further performed on phenotype analyses, including pigmentation, UV-C tolerance, virulence, growth rate, conidiation, stress (osmotic-, oxidative-, cell wall disturbing-, and high temperature-) tolerance, fungicide resistance, and conidial hydrophobicity. The results showed that the five deletion mutants (ΔPdPksP, ΔPdAbr1, ΔPdArp1, ΔPdArp2 and ΔPdAyg1) produced albino, brownish, brown, reddish-brown, and Yellowish green conidia, respectively. In addition, the survival colony forming units (CFUs) of the deletion mutants, under the treatment of UV-C radiation (261.4 mJ/cm²), were 0.3- to 0.6-fold of those surviving in wild-type strain N1. Moreover, after 522.8 mJ/cm²-UV-C-irradiation on conidia, the deletion mutants showed a larger decrease in pathogenicity on Valencia Orange fruits compared with strain N1. However, there were no significant differences among other phenotypes tested in this study. Collectively, our research reported the DHN-melanin synthesis pathway in P. digitatum for the first time, and revealed that DHN-melanin is important for P. digitatum to tolerate UV-C irradiation.     

Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of l-Tyrosine

Sporothrix schenckii is the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor of S. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally, l-dihydroxyphenylalanine (l-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism in Sporothrix spp., we cultured 73 strains, including representatives of newly described Sporothrix species of medical interest, such as S. brasiliensis, S. schenckii, and S. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. An S. schenckii DHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of the Sporothrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.     

In Vitro Interactions of Calcineurin Inhibitors with Conventional Antifungal Agents Against the Yeast Form of Penicillium marneffei

Penicillium marneffei can cause a life-threatening disseminated mycosis in immunocompromised hosts. However, therapeutic strategies for the treatment of this infectious disease are limited. Reports of other fungi suggest that calcineurin inhibitors interact with antifungal agents to improve the treatment outcomes. Here, we evaluated the in vitro interaction of the calcineurin inhibitors cyclosporine A and tacrolimus (FK506) combined with conventional antifungal agents against the pathogenic yeast form of P. marneffei. We demonstrate that the combination of cyclosporine A with amphotericin B, itraconazole, or fluconazole was synergistic for 85, 65, and 30 % of P. marneffei strains, respectively. In contrast, no synergism was observed in all the combinations containing tacrolimus. Furthermore, antagonism was not observed for any combination. In conclusion, the therapeutic potential of a combinatory approach using the calcineurin inhibitor cyclosporine A with conventional antifungal drugs may lead to improved treatment regimens for P. marneffei infections. We propose that mechanism of action studies with cyclosporine A and antifungal agents is needed.     

Detection of Talaromyces marneffei from Fresh Tissue of an Inhalational Murine Pulmonary Model Using Nested PCR

Penicilliosis marneffei, often consecutive to the aspiration of Talaromyces marneffei (Penicillium marneffei), continues to be one of the significant causes of morbidity and mortality in immunocompromised patients in endemic regions such as Southeast Asia. Improving the accuracy of diagnosing this disease would aid in reducing the mortality of associated infections. In this study, we developed a stable and reproducible murine pulmonary model that mimics human penicilliosis marneffei using a nebulizer to deliver Talaromyces marneffei (SUMS0152) conidia to the lungs of BALB/c nude mice housed in exposure chamber. Using this model, we further revealed that nested PCR was sensitive and specific for detecting Talaromyces marneffei in bronchoalveolar lavage fluid and fresh tissues. This inhalation model may provide a more representative analysis tool for studying the development of penicilliosis marneffei, in addition to revealing that nested PCR has a predictive value in reflecting pulmonary infection.     

Differentially regulated high‐affinity iron assimilation systems support growth of the various cell types in the dimorphic pathogen Talaromyces marneffei

Iron is a key trace element important for many biochemical processes and its availability varies with the environment. For human pathogenic fungi iron acquisition can be particularly problematical because host cells sequester free iron as part of the acute‐phase response to infection. Fungi rely on high‐affinity iron uptake systems, such as reductive iron assimilation (RIA) and siderophore‐mediated iron uptake (non‐RIA). These have been extensively studied in pathogenic fungi that exist outside of host cells, but much less is known for intracellular fungal pathogens. Talaromyces marneffei is a dimorphic fungal pathogen endemic to Southeast Asia. In the host T. marneffei resides within macrophages where it grows as a fission yeast. T. marneffei has genes of both iron assimilation systems as well as a paralogue of the siderophore biosynthetic gene sidA, designated sidX. Unlike other fungi, deletion of sidA or sidX resulted in cell type‐specific effects. Mutant analysis showed that T. marneffei yeast cells also employ RIA for iron acquisition, providing an additional system in this cell type that differs substantially from hyphal cells. These data illustrate the specialized iron acquisition systems used by the different cell types of a dimorphic fungal pathogen and highlight the complexity in siderophore‐biosynthetic pathways amongst fungi.     

Role of Rhizomys pruinosus as a natural animal host of Penicillium marneffei in Guangdong,China

Penicillium marneffei, a dimorphic fungus that can cause penicilliosis marneffei, is endemic in Southeast Asia. The only known hosts of P. marneffei are humans and bamboo rats. The aim of our study was to explore the distribution of P. marneffei in bamboo rats, their associated environment and non‐rat‐associated environments. Totally, 270 samples were collected in Guangdong province of China in 2012; the prevalence of P. marneffei was much higher in samples collected from surrounding areas of burrows (8.2%) than in the samples obtained from non‐rat‐associated sites (2%) or artificial farms of bamboo rats (0%). There was no difference in P. marneffei isolated rate from different areas of Guangdong province. The infection is prevalent in all rats, and this fungus could be frequently seen in the rats' lungs. This study confirms that bamboo rat is the ecological niche of P. marneffei and hypothesizes that bamboo rats become infected by inhaling aerosolized conidia originating from environmental sources, rather than by the fecal–oral route or transplacental crossing. According to the result of no detection of P. marneffei in the artificial farm, the activity of bamboo rats might be more relevant to the distribution and dissemination of P. marneffei in natural environment.     

Melanization and morphological effects on antifungal susceptibility of Penicillium marneffei

The biosynthesis of melanin has been linked with virulence in diverse pathogenic fungi. Penicillium marneffei, a dimorphic fungus, is capable of melanization in both mycelial and yeast phases, and the pigment may be produced during infection to protect the fungus from the host immune system. To investigate the impact of yeast morphological transformation on antifungal susceptibility, P. marneffei was cultured on various media including minimal medium, 1 % tryptone, brain heart infusion broth, and malt extract broth by using the standardized susceptibility protocol (the M27-A protocol, RPMI medium) for yeasts. We also investigated whether P. marneffei melanization affected its susceptibility to antifungal drugs by adding l-DOPA into culture broths. There were no differences in the minimum inhibitory concentrations of P. marneffei yeast cells previously grown in various culture broths with or without l-DOPA using the M27A protocol (into which no melanin substrate can be added due to a rapid colour change of the RPMI medium to black) for testing amphotericin B, clotrimazole, fluconazole, itraconazole and ketoconazole. However, both melanized and non-melanized P. marneffei displayed increased resistance to antifungal drugs when l-DOPA was added into a selected assay medium, 0.17 % yeast nitrogen base, 2 % glucose, and 1.5 % agar. Hence, active melanin formation appears to protect P. marneffei by enhancing its resistance to antifungal drugs.     

Role of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways in regulating replication of Penicillium marneffei in human macrophages

Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To elucidate the mechanisms involved, we investigated the role of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (p38) pathways in cytokine expression, phagosome–lysosome fusion and replication of P. marneffei in P. marneffei-infected human macrophages. Analysis of both ERK1/2 and p38 showed rapid phosphorylation in response to P. marneffei. Using specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that ERK1/2 and p38 were essential for P. marneffei-induced tumor necrosis factor-α production, whereas p38, but not that of ERK, was essential for IL-10 production. Furthermore, the presence of PD98059 always decreased phagosomal acidification and maturation and increased intracellular multiplication of P. marneffei, whereas the use of SB203580 always increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that a proper balance of between ERK1/2 and p38 may play an important role in controlling the replication of P. marneffei. Our findings further indicate a novel therapeutic avenue for treating P. marneffei by stimulating ERK1/2 or activating ERK1/2-dependent mechanisms.     

Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

Background

Penicillium marneffei is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37°C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in P. marneffei that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of P. marneffei.

Results

Whole cell proteins from the early stages of mould and yeast development in P. marneffei were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated RanA, was subsequently cloned and characterized. The P. marneffei RanA protein sequence, which contained the signature motif of Ran-GTPases, exhibited 90% homology to homologous Aspergillus proteins.

Conclusion

This study clearly demonstrates the utility of proteomic approaches to studying dimorphism in P. marneffei. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the molecular mechanisms of phase transition. Finally, the documented increased levels of RanA expression suggest that cellular development in this fungus involves additional signaling mechanisms than have been previously described in P. marneffei.     

Rapid identification and characterization of Penicillium marneffei using multiplex ligation-dependent probe amplification (MLPA) in paraffin-embedded tissue samples

Penicillium marneffei infection is a deadly disease and early diagnosis leads to prompt and appropriate antifungal therapy. To develop a sensitive method to diagnose P. marneffei infection, a multiplex ligation-dependent probe amplification (MLPA) assay was adapted. This method can rapidly and specifically detect P. marneffei DNA in cultured cells and paraffin-embedded tissue samples. Three pairs of probes were designed for amplifying the internally (intergenic) transcribed spacer (ITS) region of P. marneffei rRNA using a systematic phylogenetic analysis. These three probe sets produced three amplicons of 198, 166, and 152 bp, respectively, specific for P. marneffei. In contrast, there was only one 198 bp amplicon produced for Talaromyces stipitatus, and one 152 bp amplicon for P. funiculosum, T. intermedius and T. derxii. The probes did not amplify any other reference strains. An array of 40 P. marneffei strains isolated from human patients, bamboo rat, and the local environment was tested by using MLPA, and all were positively identified. Most importantly, P. marneffei in paraffin-embedded tissue specimens from infected human patients was positively amplified by MLPA. The sensitivity and specificity of the MLPA assay could be a useful tool for prompt diagnosis, pathogen characterization, and epidemiological studies of fungal infections.     

1,8-Dihydroxynaphthalene (DHN)-Melanin Biosynthesis Inhibitors Increase Erythritol Production in Torula corallina, and DHN-Melanin Inhibits Erythrose Reductase

The yeast Torula corallina is a strong erythritol producer that is used in the industrial production of erythritol. However, melanin accumulation during culture represents a serious problem for the purification of erythritol from the fermentation broth. Melanin biosynthesis inhibitors such as 3,4-dihydroxyphenylalanine and 1,8-dihydroxynaphthalene (DHN)-melanin inhibitors were added to the T. corallina cultures. Only the DHN-melanin inhibitors showed an effect on melanin production, which suggests that the melanin formed during the culturing of T. corallina is derived from DHN. This finding was confirmed by the detection of a shunt product of the pentaketide pathway, flaviolin, and elemental analysis. Among the DHN-melanin inhibitors, tricyclazole was the most effective. Supplementation with tricyclazole enhanced the production of erythritol while significantly inhibiting the production of DHN-melanin and DHN-melanin biosynthetic enzymes, such as trihydroxynaphthalene reductase. The erythrose reductase from T. corallina was purified to homogeneity by ion-exchange and affinity chromatography. Purified erythrose reductase was significantly inhibited in vitro in a noncompetitive manner by elevated levels of DHN-melanin. In contrast, the level of erythrose reductase activity was unaffected by increasing concentrations of tricyclazole. These results suggest that supplemental tricyclazole reduces the production of DHN-melanin, which may lead to a reduction in the inhibition of erythrose reductase and a higher yield of erythritol. This is the first report to demonstrate that melanin biosynthesis inhibitors increase the production of a sugar alcohol in T. corallina.